1. Field of the Invention
The present invention relates to methods, systems and kits for fast and enzymeless cloning of nucleic acid fragments into vectors and for forced cloning selection for successful transformation.
2. Description of the Related Art
Traditional molecular cloning involves the use of recombinant DNA technology to propagate DNA fragments inside a foreign host. Generally, the DNA fragments are isolated from cDNA libraries or chromosomes and subcloned into a vector utilizing various enzymes. For example, a small amount (i.e., 0.01-0.03 μg) of isolated DNA fragment is contacted with a small amount (i.e., 0.01 μg) of linearized vector. Using enzymes, such as ligases, the fragments are ligated.
The DNA fragment-containing vector is introduced into a host cell according to various methods of transformation. For example, one tenth to one half of the ligation mix can be electroporated into a cell, such as E. Coli. Generally, a large number of cells, such as 1×108, is used to increase the ratio of cells to DNA fragment-containing vector to enhance the probability of obtaining a cell with the desired clone. For example, the ration might be 0.02-0.2 fg/cell.
A selection marker is usually included in the vector to increase the probability that the host cell has the DNA fragment-containing vector. Following introduction into the host cell and selection of the host cell containing the vector, the DNA fragment within the vector can then be replicated along with the host cell DNA. The DNA fragment-containing vector then can be isolated and purified from the host cell and transfected into animal cells or tissues for functional analysis of the encoded gene product.
Although the traditional enzymatic cloning methods have advantages such as pinpoint accuracy, they also have significant drawbacks. As mentioned, the methods require the use various enzymes that can be very expensive. In addition, the same DNA fragment has to be enzymatically treated every time it is introduced into a different vector. All of the vector may not be effectively cut by the enzymes, which can result in a higher number of background cells. Also, the methods involve slow and laborious processes. Selection of host cells containing the DNA fragment-containing vector entails significant labor and is still an uncertain process. Traditional cloning methods, even in conjunction with the use of polymerase chain reaction (PCR), are still time consuming, costly and difficult to automate.
The present invention provides simple and rapid methods, systems and kits for cloning nucleic acid fragments.